1. Codon Usage Determines the Mutational Robustness, Evolutionary Capacity, and Virulence of an RNA Virus 
Adam S. Lauring, Ashley Acevedo, Samantha B. Cooper, Raul Andino 
Cell Host & Microbe 
Volume 12, Issue 5, Pages (November 2012)
DOI: /j.chom
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2. Figure 1
Using Synonymous Mutation to Place Viral Populations in Unique Regions of Sequence Space with Distinct Fitness Landscapes
(A) Schematic of codon networks and their hypothesized role in viral evolution. In this “pipe” network (top), connections represent evolutionary pathways. Coloring is according to hypothetical fitness values for the corresponding viral variant (red, highest; blue, lowest; gray, zero fitness). The bottom panel shows two variants with distinct mutant neighborhoods. While these variants are connected by a neutral, synonymous mutation, they produce progeny with distinct fitness values and adaptive capacity. Because large-scale synonymous mutation places populations in distinct regions of sequence space, the networks are hypothesized to have different genetic structures, which will affect the evolutionary capacity of the population (adapted from van Nimwegen [2006], Draghi et al. [2010]).
(B) Schematic of synthetic viruses used in this study. The genomic organization of poliovirus type 1 (PV1, WT) is shown. All viral proteins are derived by proteolytic cleavage of a single polyprotein. The synthetic viruses Max and SD were designed as negative controls for studies of codon usage and polyprotein translation. Each has hundreds of synonymous substitutions across the capsid sequence relative to the WT, while preserving overall codon bias and GC content. Max has codon pair bias matched closely to that of the human genome. In SD, the codons are shuffled randomly throughout the capsid coding region.
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3. Figure 2 Synonymous Populations Differ in Their Robustness to Mutation
(A) Fitness distribution of variants in virus populations. Histograms of plaque size for WT (hatched bars, both panels, n = 1,053), Max (white bars, top panel, n = 1,146), and SD (white bars, bottom panel, n = 937).
(B) Synonymous populations exhibit differential sensitivity to a mutagenic nucleoside. The titers of ribavirin-treated populations were normalized to mock-treated controls. Data for WT (filled circles), Max (open squares), SD (open triangles), and G64S (ribavirin resistant variant, asterisks) populations are shown. Data points and error bars represent mean ± 95% confidence interval for five replicates, and significant differences between WT and SD are indicated (t test, ∗p < 0.05).
(C) Reduction in plaque size in ribavirin-treated clones. Each population was passed in 400 μM ribavirin (+R) or mock-treated media for 24 hr. Plaques from each population were quantified as in (b). Box plots indicate median titer, 25% and 75% quartiles, and 1.5 ∗ interquartile range.
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4. Figure 3 Synonymous Mutation Affects the Fitness of Virus Populations
(A) Population fitness as measured by direct competition. The amount of each virus at each passage was compared to the input. Data are expressed as the log10 change in ratio for Max (open squares) and SD (open triangles) over time relative to WT (filled circles, shown here as reference). The slope reflects the relative fitness of each population. Representative data are shown and values for mean ± SD, n = 5, are given in the text.
(B) Kinetics of genome replication for WT (filled circles), Max (open squares), and SD (open triangles) with five replicates for each virus and time point. Mean ± bootstrap-derived standard deviation for key parameters include exponential growth rate (WT  ± , Max  ± , SD  ± ), maximum genome number (WT  ± , Max  ± , SD  ± ), and area under the curve (WT  ± , Max  ± , SD  ± ). See also Figure S1.
(C) Protein expression in radiolabeled, infected cells. Capsid proteins (VP0-VP3) were identified by comparison to an anticapsid immunoprecipitate, left.
(D) Quantitation of capsid gel bands for WT (dark gray), Max (light gray), and SD (white). Mean ± SD, n = 5.
(E) Changes in fitness at elevated mutation rates. WT (filled circle), Max (open square), and SD (open triangle) populations were competed against isogenic, ribavirin-resistant references containing a single point mutation, 3DG64S. Competition assays were performed over seven single-cycle, low-moi (<0.5) passages. Shown are fitness values (mean ± SD, n = 4) at varying ribavirin concentration.
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5. Figure 4 Deep Sequencing of Mutant Spectra
(A) Variants in WT, Max, and SD were cloned by endpoint dilution, and 400–500 clones were pooled and sequenced. The cloning process shifted the frequency of observed SNVs, reducing the likelihood of false-positive base calls. Variants with a frequency >0.001 were used in downstream analyses. Two replicate pools of clones were sequenced for a total of ∼850 clones for each virus. See also Figures S2 and S4 and Table S1.
(B) Pie charts show the number of unique variants identified in the 5′ noncoding region (5′UTR), capsid protein (P1), nonstructural proteins (P2/P3), and 3′ noncoding region (3′UTR). The numbers of nonsynonymous (NS) and synonymous (Syn) substitutions in coding regions are also indicated.
(C) Venn diagrams show overlap in mutant nucleotide position in clones derived from each population.
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6. Figure 5
Synonymous Populations Exhibit Distinct Sets of Polymorphic Amino Acid Substitutions
An amino acid alignment of enterovirus capsids was used to derive a variability score for each codon (top). Higher values indicate more observed variation (Valdar, 2002). Arrows highlight antigenic regions of poliovirus capsid. Heatmap (bottom) of location and blosum62 conservation score for each codon (Henikoff and Henikoff, 1992). Both nonsynonymous and synonymous substitutions are included, and invariant amino acids are shown in black. For codons with ≥2 distinct variants, the less-conserved substitution (lower blosum62 score) is shown. See also Figures S3 and S4.
Cell Host & Microbe  , DOI: ( /j.chom )
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7. Figure 6
Mutational Robustness of Poliovirus Populations Is Strongly Correlated with Virulence in a Mouse Model of Infection
(A) Survival analysis of poliovirus receptor transgenic mice infected intramuscularly with WT (solid line), Max (dotted line), or SD (dashed line) populations, n = 25 mice per group.
(B) Titer of virus recovered from brains of mice 5 days after intravenous inoculation. Box plots indicate median titer, 25% and 75% quartiles, and 1.5 ∗ interquartile range for each virus population, n = 10 mice per group.
(C) Sequence analysis of brain-derived mutant spectra. For each virus, ∼540 clones were recovered from the brains of ten infected mice and pooled for deep sequencing. Histograms show the frequency of SNVs identified in each pool of clones. See also Table S1.
Cell Host & Microbe  , DOI: ( /j.chom )
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